Cyclic dipeptides (CDPs) are amino acid-based compounds, some of which possess antibacterial activity. The encapsulation of certain drugs into liposomes has been found to improve their activity in terms of bioavailability and duration of action. Liposomes are small vesicles that are under investigation as drug carriers for the delivery of therapeutic agents. A number of liposome formulations are currently under clinical trial review, whilst some have already been approved for clinical use. The aim of this study was to optimize a liposomal cyclo(Tyr-Pro) formulation and to assess its antibacterial activity against various Gram-positive and Gram-negative bacteria. Response surface methodology (RSM) using the central composite design (CCD) model was used to optimize liposomal formulations of cyclo(Tyr-Pro) for each of the four bacteria, namely Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Percent drug encapsulated and bacterial inhibition were investigated with respect to two independent variables, i.e. lipid composition and cholesterol content. Design Expert 8 was used for the purpose of finding the combination of independent variables that would yield an optimal formulation for each bacterium. The model selected by the software failed to adequately correlate the predicted models to the experimental data. The in vitro experiments showed that the antibacterial activity of liposome-encapsulated cyclo(Tyr-Pro) was superior to that of its free counterpart. Binding maximum or Bmax for the encapsulated compound at concentrations as low as 0.412 mg/ml, was significantly higher than that obtained for free cyclo(Tyr-Pro) which was tested at a concentration of 20 mg/ml. Furthermore, encapsulation of cyclo(Tyr-Pro) into a liposome formulation enhanced its potency. This was evident in the lower IC50 values for the liposomal compound when compared to free cyclo(Tyr-Pro).

No description available.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Antimicrobial peptides (AMPs) are currently the most researched group of compounds for new antimicrobial drugs especially with the rise in resistance to almost all available drugs by public health relevant pathogens. In this study we set out to characterise small cyclic AMPs in terms of their activity towards human pathogens Listeria monocytogenes, a food-borne pathogen causing listeriosis and Plasmodium falciparum, a parasite that causes malaria respectively, each a threat to public health. One of the small cyclic peptide libraries examined is the tyrocidines (Trcs) and analogues, which are cyclic decapeptides [cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu] produced by the Gram-positive bacteria Bacillus aneurinolyticus as part of the tyrothricin complex which is non-ribosomally synthesised during sporulation. Previous research found that the six major Trcs were active against Listeria monocytogenes and Plasmodium falciparum and it was found that the identity of the aromatic residues in the aromatic dipeptide unit has an important role in activity. We set out to extend the qualitative structure to activity relationship (QSAR) studies using more Trc analogues and small synthetic Arg- and Trp-rich cyclic peptides (RW-peptides) in a bid to establish essential structural motifs and pre-requisites for activity. Eight natural and three synthetic Trc analogues and fifteen RW-peptides were either naturally or by chemical synthesis produced and characterised in terms of chemical character and biological activity. The Trcs were significantly more active than RW peptides, although much more haemolytic and thus toxic. Results indicated the relevance for hydrogen bonding with an aromatic amino acid residue for selective activity towards the leucocin A resistant L. monocytogenes B73-MR1. However, structural properties favouring a tighter membrane interaction hindered the Trc mode of action (MOA). We determined that Gln6 and hydroxyl group of Tyr7 may be involved in interaction with the putative target in L. monocytogenes. There was also need for an amphipathic balance between hydrophobicity and size/steric parameters for optimal activity. From our QSAR studies we predict as lead peptide for a future library of antilisterial Trcs: cyclo(VOMe3LfPWfNQY). Furthermore, the antilisterial activity of the Trcs was found to be predominantly lytic and salt tolerant while RW-peptides were non-lytic and sensitive to Ca2+. We confirmed that Ca2+ enhanced Trc antilisterial activity with Ca2+ increasing the Trc anti-metabolic activity, but conversely inducing a non-lytic mechanism of action. From model membrane studies, we propose that the calcium induced Trc non-lytic MOA could be due to detrimental lipid demixing, presence of a Trc sensitive Ca2+-induced non-membrane target in the prematurely calcium induced intracellular anaerobic form of Listeria monocytogenes, and/or the Trc-Ca2+ complexes may inhibit key components such as membrane bound electron transport system or bacterial dehydrogenases. We confirmed, as previously found, that the Trcs have potent antimalarial activity that is sequence specific and non-lytic. The RW-peptides had very weak activity, but our results again indicated that more hydrophobic and haemolytic peptides tend to be more active, particularly the RW-peptide containing the Trp analogue β-(benzothien-3-yl)-alanine (Bal). A novel finding was that one of the more polar Trc C analogues, namely tryptocidine C (Tpc C), in contrast to Trc C showed potent antimalarial activity indicating the specific sequence and the role of the Trp7 in activity. From these results a proposed lead peptide for future research is cyclo[VOLfP(Bal)fNQ(Bal)]. Furthermore, in our search for the Trc and Tpc C target(s) we employed high resolution fluorescence microscopy. Results show that Trc led to disorganisation of neutral lipid structures and chromatin halting growth in late trophozoite/early schizont stages. This indicated that membrane structures containing neutral lipids, as well as chromatin may be targeted by the Trcs. Another novel finding in our studies was that chloroquine (CQ) resistance not only correlated with resistance to Trcs, but the Trcs and CQ were found to be antagonistic towards each other’s activity. This indicated a shared target and we propose the food vacuole as another of the Trc targets in P. falciparum. / AFRIKAANSE OPSOMMING: Antimikrobiese peptiede (AMPe) is tans die mees nagevorsde groep verbindings in die soeke na nuwe antimikrobiese middels, veral weens 'n toenemende weerstandigheid van patogene in die openbare gesondheidsektor teen alle beskikbare middels. Die doel van hierdie studie was om klein, sikliese AMPe in terme van hul aktiwiteit teenoor twee menslike patogene wat 'n bedreiging vir openbare gesondheid is, Listeria monocytogenes, 'n voedsel-oordraagbare patogeen wat listeriose veroorsaak, asook Plasmodium falciparum, die parasiet verantwoordelik vir malaria, te karakteriseer. Een van die klein, sikliese peptiedbiblioteke wat ondersoek is, is die tyrocidines (Trcs) en analoë (sikliese dekapeptiede [siklo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu]). Hierdie peptiede deur die Gram-positiewe bakterie Bacillus aneurinolyticus word wat nie-ribosomaal gesintetiseer as deel van die tirotrisien kompleks word tydens sporulasie. Vorige navorsing het gewys dat die ses hoof Trcs teen Listeria monocytogenes en Plasmodium falciparum aktief is en dat die identiteit van die aromatiese residue in die aromatiese dipeptiedeenheid 'n belangrike rol speel in die Trc-aktiwiteit. Ons het gepoog om die kwalitatiewe struktuur-aktiwiteit-verwantskap (QSAR) studies uit te brei deur meer Trc analoë en klein sintetiese Arg- en Trp-ryke sikliese peptiede (RW-peptiede) te gebruik en sodoende essensiële struktuur-motiewe en voorvereistes vir aktiwiteit vas te stel. Agt natuurlike en drie sintetiese Trc analoë, asook vyftien RW-peptiede is of deur natuurlike of chemiese sintese geproduseer en gekarakteriseer in terme van chemiese karakter en biologiese aktiwiteit. Die Trcs het beduidend meer aktiwiteit as RW-peptiede getoon, maar is ook meer hemolities en dus meer toksies. Die resultate dui op die belang van waterstofbinding met 'n aromatiese aminosuurresidu vir die selektiewe aktiwiteit teenoor die leucocin A weerstandige L. monocytogenes B73-MR1. Strukturele eienskappe wat tot 'n sterker membraan-interaksie lei, verhinder egter die werkingsmeganisme. Ons het vasgestel dat Gln en die hidroksielgroep van Tyr betrokke kan wees in die interaksie met die vermeende teenmiddelteiken in L. monocytogenes. 'n Balans tussen amfipatiese/hidrofobiese en grootte/steriese parameters is ook noodsaaklik vir optimale aktiwiteit. Vanuit ons QSAR studies word die peptied siklo-(VOMe3LfPWfNQY) as die voorloper vir 'n toekomstige peptiedbiblioteek van antilisteriale Trcs voorgestel. Verder is daar gevind dat die antilisteriese aktiwiteit van die Trcs oorwegend lities en sout-verdraagsaam is, terwyl die RW-peptiede nie-lities en Ca2+ sensitief is. Ons het bevestig dat Ca2+ die Trc antilisteriese aktiwiteit verbeter, deur die Trc se antimetaboliese aktiwiteit verhoog, maar terselfdertyd 'n nie-litiese werkingsmeganisme induseer. Vanuit model-membraan studies word voorgestel dat Trc se nie-litiese werkingsmeganisme, soos teweeggebring deur Ca2+, die gevolg kan wees van nadelige lipied vermenging, die teenwoordigheid van 'n kalsium geïnduseerde Trcsensitiewe nie-membraan teiken in 'n vervroegde kalsium geïnduseerde intrasellulêre anaerobiese vorm van Listeria monocytogenes, en/of dat die Trc-Ca2+ komplekse belangrike komponente soos ’n membraan-gebonde elektron transport sisteem of bakteriële dehidrogenases inhibeer. Daar is ook bevestig, soos voorheen gevind, dat die Trcs kragtige, antimalaria aktiwiteit besit wat volgorde-spesifiek en nie-lities is. Die RW-peptiede het swak aktiwiteit getoon, maar ons resultate het weereens bewys dat peptiede wat meer hidrofobies en hemolities is, meer aktief is, veral die RW-peptiede wat die Trp analoog β-(bensoteïen-3-iel)-alanien (Bal) bevat. 'n Nuwe bevinding is dat een van die meer polêre Trc C analoë, genaamd triptosidien C (Tpc C), in teenstelling met Trc C, sterk antimalaria aktiwiteit het, wat 'n aanduiding is van die spesifieke volgorde en die rol van die Trp7 in aktiwiteit. Vanuit hierdie bevindinge word die peptied siklo- (VOLfP(Bal)fNQ(Bal)) as 'n voorloper vir toekomstige navorsing aangedui. Vir ons soeke na die Trc en Tpc C teiken(s), het ons hoë resolusie fluoressensie mikroskopie aangewend. Resultate toon dat Trc tot die ontwrigting van 'n neutrale lipied strukture en chromatien lei en sodoende groei beperk in die laat trofosoïet/vroeë skisont fases. Dit het aangedui dat die membraanstrukture wat neutrale lipiede bevat, sowel as chromatien, deur die Trcs geteiken word. 'n Verdere nuwe bevinding in hierdie studie was dat chloroquine (CQ) weerstandigheid nie net korreleer met weerstandigheid teen Trcs nie, maar dat die Trcs en CQ antagonisties optree teenoor mekaar se aktiwiteite. Dit dui op 'n gemeenskaplike teiken en die kosvakuool as 'n addisionele Trc teiken in P. falciparum word voorgestel.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

upd3 in the haemocytes also caused a reduction to signalling in the JNK pathway. The results here show that the haemocytes relay signal(s) to the fat body through the use of cytokines, a process surprisingly similar to the mammalian system.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Insects are confronted by a large variety of potentially harmful microorganisms to which they are resistant as they are able to build up an efficient innate defense system that relies on three tightly interconnected reactions. One of the reactions is the transient and rapid synthesis of a battery of antimicrobial peptides (AMPs). Development of antimicrobial therapeutic drugs and vaccines is very crucial due to factors such as the emergence of multiple-drug resistance. AMPs have been termed natural antibiotics because of their large spectrum of activity. The current study focused on the isolation and identification of cationic antimicrobial peptides and the analysis of immune response in the South African Euoniticellus intermedius embryonic (SAEIE08) cell line upon exposure to pathogens. E. intermedius is of the Coleopteran order in the Scarabaeoidea superfamily. Liquid growth inhibition assay showed higher antimicrobial activity in SAEIE08 that was treated with heat-killed E. coli compared to untreated. Further evidence for antimicrobial activity was seen as a clear zone of inhibition in solid growth inhibition assay when a gel run with protein extracts was plated and overlayed with live E. coli. Changes in protein expression patterns that were analysed in SDS-PAGE and 2-D PAGE indicated the most intense bands and spots at low molecular weight sizes around 10 kDa and/or 16 kDa which implicated increased induction of AMP expression upon exposure to pathogen. Homologues of Saccharomyces cerevisiae proteins were found in some of the 5′/3′ RACE sequences. Possible explanation for matches to these homologues could be that short sequences were used for database searches. The proteins were identified as flavin-containing monooxygenase, long-chain fatty acyl-CoA synthetase, severe depolymerization of actin protein and serine/threonine protein kinase. Interestingly, these proteins play roles in metabolism, cell proliferation and/or molecular pathways which do occur when cells are exposed to stress. There was also an insect peptide allatotropin from Spodoptera frugiperda. The results show that there is inducible antimicrobial activity in embryonic E. intermedius cell line.

Thesis (Master of Applied Sciences in Chemistry)--Cape Peninsula University of Technology, 2018. / Antimicrobial Peptides (AMPs) are short peptides of about 45 - 54 amino acids that exhibit antibacterial and antifungal activities. Plant defensin is a type of AMP in plants which belong to a family of cationic peptides with a characteristic 3D folding pattern held in place by four disulfide bridges. AMPs especially defensins have been identified to have a huge biotechnological potential and are being patented for many applications. The aim of this work was to clone an antimicrobial peptide from Vigna subterranea and characterise it with bioinformatics analysis. 4 sets of primers were synthesized according to the sequences of conserved regions in AMPs i.e. defensins from legumes like Vigna unguiculata, Vigna radiata, Cicer arietinum and Cajanus cajan, amongst others, which have defensins with only a few sequence differences. The primers were designated VsDef P1 to P4. Using Vigna subterranea total genomic DNA as a template, fragments of expected sizes were successfully amplified and cloned into the pDRIVE vector and used to transform Escherichia coli JM109 cells in each case. Representative clones were sequenced and analysed using BLAST from National Center for Biotechnology Information. However, only the VIG clone was shown to be a bona fide defensin (over 90% identity, E-value of 1ex102, 99% query coverage of the nucleotide sequence, compared to Vigna unguiculata defensin). Based on this high sequence identity, a new pair of primers VsDef P5 was designed based on the Vigna unguiculata defensin sequence to specifically amplify the complete Vigna subterranea defensin gene, hereafter called VsDef1. Attempts to clone VsDef1 were however unsuccessful, and evidence of clone deletion and insert re-arrangement of insert DNA was observed. Direct sequencing of the PCR product demonstrated that it was indeed the complete VsDef1 pre-protein, composed of 433 nucleotides. In silico translation and analysis showed that VsDef1 has an intron at position 105 − 259 of the nucleotide sequences and encodes for a 78 amino acid peptide. Phylogenetic analysis revealed to be similar to the sequence of the defensins for Vigna unguiculata (96%), Vigna radiata (95%), Vigna angularis (95%) and Phaseolus vulgaris (93%) on the NCBI database. The three - dimensional structure of the peptide was modelled with SWISS-MODEL expasy and the structure was found to include one α- and three β domains, similar to those of other defensins. The failure to identify VsDef1 clone in a V. subterranea library and the failure to recover its cDNA clone are consistent with the hypothesised toxicity of VsDef1 to Escherichia coli. It is suggested that a different host, such as yeast, should be used in the future. The VsDef1 mRNA levels in germinating V. subterranea seeds was however successfully investigated using real-time reverse transcription quantitative PCR. VsDef1 mRNA is present in both the testa and embryo of dry seed and will persist through the early stages of seedling growth. This demonstrates the importance of VsDef1 in fighting off infection during germination in order to ensure successful germination. It is therefore essential to characterise more antimicrobial peptides from V. subterranea. The diversity of AMPs and their patterns of expressed genes will enable understanding of complex regulatory networks, which will likely enable identifying of genes involved in diseases and new biological processes.

宿主防御肽，如抗菌肽和防御素，是固有性免疫的重要组成部分。LL-37是由37个残基组成的阳离子宿主防御肽，是目前唯一被发现的人源宿主防御肽。它在不同的生物过程中都起着关键作用。新证据表明，LL-37与肿瘤进展也有关系。在许多类型的人类恶性肿瘤中它有不同表达，但在结肠癌中的表达和作用，仍未知。在此，我们将对LL-37及其17至32残基片断（ 简称FK-16）对结肠癌的影响进行研究。 / 免疫组化染色结果表明，LL-37在人类结肠癌组织中的表达比正常组织有显著减少。并且，LL-37的表达与TUNEL阳性细胞数量呈正比。合成的LL-37能够诱导不同的结肠癌细胞发生不依赖半胱天冬酶激活的凋亡细胞死亡。并且，LL-37通过激活p53下调Bcl2及上调Bax与Bak来诱导凋亡。LL-37也促使肿瘤坏死因子和核酸内切酶G 向核内转移，以其为目标的siRNA沉默能使细胞对LL-37诱导的凋亡呈现出耐受现象。更重要的是，LL-37的促凋亡作用被发现可以通过对百日咳敏感的Gαi蛋白偶联受体来介导。同时，宿主防御肽敲除的小鼠肠黏膜中，p53、Bax和Bak表达减少而Bcl2表达增加，凋亡的基础水平量也减少。由此说明，LL-37可通过激活GPCR-p53-Bax / Bak / Bcl-2的新信号级联反应来激活AIF / EndoG调控的结肠癌细胞凋亡。 / 与LL-37类似，其片断FK-16也促使不同结肠癌细胞株死亡。但其死亡诱导机制与LL-37不尽相同。FK-16引发了一种独特的死亡方式，即初始诱导不依赖半胱天冬酶激活的凋亡之后紧随引发自噬性细胞死亡。而LL-37没有明显引起这种自噬性死亡。孵育FK-16 24小时后，结肠癌细胞被证明发生凋亡。延长孵育至48小时，细胞的生化和形态学体征符合自噬，包括增加LC3阳性自噬体，积累酸性自噬泡与自溶酶体，和提高 LC3-II水平。敲除两个自噬有关基因 ATG5 和ATG7, 能够部分逆转由FK-16所引起的细胞死亡。并且，细胞凋亡和细胞自噬机制相关信号通路之间存在的交叉调控，在此研究中也被深入提及。 / Host defense peptides, such as cathelicidins (LL-37) and defensins, are important components in the innate immunity. LL-37, a human cationic host defense peptide composed of 37 residues, is the only cathelicidin described so far in humans. It plays a key role in diverse biological processes, including natural immunity, inflammation and tissue repair. Emerging evidence suggests that LL-37 is implicated in cancer development. In this regard, the expression of LL-37 is found to be dysregulated in many types of human malignancy, including lung, breast, ovarian, and gastric cancers. The expression and function of LL-37 in colon cancer, however, are still unknown. In this thesis, the roles of LL-37 and its 17-32 fragment (hereafter referred to as FK-16) in colon cancer development were investigated. / By immunohistochemical staining, it is demonstrated that the expression of LL-37 was significantly reduced in human colon cancer tissues as compared with the cancer adjacent normal tissues. Moreover, LL-37 expression was positively correlated with the number of TUNEL-positive cells. Furthermore, synthetic LL-37 induced caspase-independent apoptotic cell death in different cultured colon cancer cells. In this connection, LL-37 induced apoptosis via downregulation of Bcl-2 and upregulation of Bak and Bax in a p53-dependent manner. It also induced the upregulation and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG), whose targetings by siRNAs rendered the cells resistant to LL-37-induced apoptosis. Above all, the pro-apoptotic effect of LL-37 was found to be mediated through a pertussis toxin-sensitive Gαi protein-coupled receptor. Concordantly, colonic mucosa of cathelicidin-knockout mice exhibited reduced expression of p53, Bax and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Taken together, we demonstrated that LL-37 activates a novel signaling cascade involving the GPCR-p53-Bax/Bak/Bcl-2 axis to activate AIF/EndoG-mediated apoptosis in colon cancer cells. / Similar to the effect of LL-37 peptide, the fragment FK-16 also induced cell death in colon cancer cell lines. However, the action is different. Results demonstrated that FK-16 triggered a unique pattern of cell death characterized by initial caspase-independent apoptosis followed by autophagic cell death, the latter of which was not observed obviously in cells treated with LL-37. Treating colon cancer cells with FK-16 for 24 h induced apoptosis as evidenced by phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Prolonged treatment with FK-16 induced biochemical and morphological features consistent with autophagy, including increased formation of LC3+ autophagosomes, the accumulation of acidic vesicular organelles and autolysosomes, and increased levels of LC3-I/II, Atg5 and Atg7. Knockdown of Atg5 or Atg7 partially reversed the cytotoxic effect of FK-16, suggesting that FK-16-induced autophagy was pro-death in nature. Furthermore, the novel cross-talks between apoptotic and autophagic signalings were also noted. / Collectively, the present study not only contributes to understanding the role of host defense peptide cathelicidin in tumorigenesis, but also provides pre-clinical evidence to propel the development and application of these peptides as novel therapeutic agents for the treatment of colon cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / 综上所述，目前的研究不仅有助于理解宿主防御肽在肿瘤发生, 同时也提供了临床前研究证据，推动了宿主防御肽的开发和应用, 这些肽片段为治疗结肠癌提供了新的治疗手段 。 / Ren, Shunxiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 176-208). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Declaration --- p.vi / Acknowledgements --- p.vii / Publications --- p.ix / Table of contents --- p.xiii / List of illustrations --- p.xviii / Abbreviations --- p.xxiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Colorectal cancer --- p.1 / Chapter 1.1.1 --- Epidemiology of colorectal cancer --- p.1 / Chapter 1.1.2 --- Etiology of colorectal cancer --- p.3 / Chapter 1.1.3 --- Pathogenesis of CRC --- p.8 / Chapter 1.1.4 --- Chemotherapy of colorectal cancer --- p.9 / Chapter 1.2 --- Programmed cell death (PCD) --- p.10 / Chapter 1.2.1 --- Cell death --- p.10 / Chapter 1.2.2 --- Apoptosis --- p.11 / Chapter 1.2.2.1 --- Mechanisms of apoptosis --- p.12 / Chapter 1.2.2.1.1 --- Caspase-dependent apoptosis --- p.13 / Chapter 1.2.2.1.2 --- Caspase-independent apoptosis --- p.15 / Chapter 1.2.2.1.3 --- Tumor suppressor p53 --- p.17 / Chapter 1.2.2.2 --- G-coupled protein receptors in apoptosis --- p.18 / Chapter 1.2.3.1 --- Types of Autophagy --- p.20 / Chapter 1.2.3.2 --- Biological process --- p.22 / Chapter 1.2.3.3 --- Biological functions --- p.24 / Chapter 1.2.3.4 --- Autophagic machinery --- p.27 / Chapter 1.2.3.5 --- Autophagy in cancer --- p.30 / Chapter 1.2.3.6 --- Autophagy and apoptosis --- p.32 / Chapter 1.3 --- Biological functions of cathelicidin --- p.33 / Chapter 1.3.1 --- Antimicrobial activity --- p.34 / Chapter 1.3.2 --- Immunological functions --- p.35 / Chapter 1.3.3 --- Wound healing, angiogenesis and mitogenesis --- p.36 / Chapter 1.3.4 --- Programmed cell death --- p.38 / Chapter 1.4 --- Aim of the present study --- p.39 / Chapter Chapter 2 --- Methods / Chapter 2.1 --- General --- p.41 / Chapter 2.1.1 --- Chemicals and reagents --- p.41 / Chapter 2.1.2 --- Antibodies --- p.44 / Chapter 2.1.3 --- Commercial kits --- p.45 / Chapter 2.1.4 --- Peptide synthesis --- p.46 / Chapter 2.1.5 --- Experimental Animals --- p.46 / Chapter 2.1.6 --- Cell Culture --- p.47 / Chapter 2.2 --- DNA Methylation Analysis --- p.48 / Chapter 2.2.1 --- 5-aza-2’-deoxycytidine (5’Aza-dC) Treatment --- p.48 / Chapter 2.2.2 --- Bisulfite Genomic Sequencing and Methylated-DNA capture (MethylCap)-qPCR --- p.48 / Chapter 2.3 --- Effects of LL-37 and its analogue FK-16 in colon cancer cells in vitro --- p.48 / Chapter 2.3.1 --- Cell viability Assay --- p.49 / Chapter 2.3.2 --- Lactic dehydrogenase (LDH) activity --- p.49 / Chapter 2.3.3 --- Cell cycle analysis --- p.49 / Chapter 2.3.4 --- Measurement of apoptosis in vitro --- p.50 / Chapter 2.3.4.1 --- Quantitation of DNA fragmentation --- p.50 / Chapter 2.3.4.2 --- Quantitation of phosphatidylserine externalization --- p.50 / Chapter 2.3.5 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.3.6 --- Nuclear protein extraction --- p.52 / Chapter 2.3.7 --- Western Blot --- p.53 / Chapter 2.3.8 --- Immunofluorescence --- p.54 / Chapter 2.3.9 --- Bcl-2 overexpression --- p.54 / Chapter 2.3.9.1 --- Transforming competent cells --- p.55 / Chapter 2.3.9.2 --- Plasmid DNA purification --- p.55 / Chapter 2.3.10 --- RNA interference --- p.56 / Chapter 2.3.11 --- Detection of acidic vesicular organelles (AVOs) with acridine orange --- p.57 / Chapter 2.3.12 --- Labeling of autophagic vacuoles with monodansylcadaverine (MDC) --- p.58 / Chapter 2.3.13 --- Transmission electron microscopy --- p.59 / Chapter 2.4 --- Cathelicidin-knockout (Cnlp/) mice model --- p.60 / Chapter 2.4.1 --- Normal mouse colon sample collection --- p.60 / Chapter 2.4.2 --- Tissue Processing --- p.61 / Chapter 2.4.3 --- Measurement of basal apoptosis in normal colon tissues --- p.61 / Chapter 2.5 --- Clinical samples --- p.62 / Chapter 2.5.1 --- Immunohistochemistry of clinical samples --- p.62 / Chapter 2.5.2 --- Measurement of colonocyte apoptosis of clinical samples --- p.62 / Chapter 2.5.3 --- Evaluation of colonocyte proliferation of clinical samples --- p.63 / Chapter 2.6 --- Statistical analysis --- p.63 / Chapter Chapter 3 --- Results and Discussion / Chapter 3.1 --- LL-37 was downregulated in colon cancer tissues --- p.64 / Chapter 3.2 --- Effects of LL-37 on human colon cancer cells --- p.72 / Chapter 3.2.1 --- LL-37 induced DNA fragmentation and phosphatidylserine externalization without caspase activation in colon cancer cells --- p.72 / Chapter 3.2.2 --- LL-37 induced AIF- and EndoG-dependent apoptosis --- p.79 / Chapter 3.2.3 --- Altered expression of Bcl-2 family members was required for AIF- and EndoG-mediated apoptosis induced by LL-37 --- p.84 / Chapter 3.2.4 --- p53 activation was required for LL-37-induced apoptosis --- p.90 / Chapter 3.2.5 --- The apoptogenic action of LL-37 was mediated by G protein-coupled receptor (GPCR) --- p.94 / Chapter 3.2.6 --- Reduced basal apoptotic rate in colonic mucosa of cathelicidin-knockout mice --- p.95 / Chapter 3.2.7 --- Preliminary Discussion --- p.103 / Chapter 3.3 --- Effects of FK16 on human cancer cells --- p.108 / Chapter 3.3.1 --- FK-16 induced AIF- and EndoG-dependent apoptosis in colon cancer cells --- p.108 / Chapter 3.3.2 --- FK-16 induced autophagic cell death in colon cancer cells --- p.116 / Chapter 3.3.3 --- Activation of p53 was required for FK-16-indcued apoptosis and autophagy cell death --- p.123 / Chapter 3.3.4 --- Altered expression of Bcl-2 and Bax was required for FK-16-indcued apoptosis and autophagic cell death --- p.129 / Chapter 3.3.5 --- FK-16-induced apoptosis and autophagic cell death were reciprocally regulated --- p.134 / Chapter 3.3.6 --- Preliminary Discussion --- p.139 / Chapter Chapter 4 --- Summary and Finial Conslusion --- p.142 / References --- p.144

Antimicrobial peptides are active against bacteria, fungi and viruses as part of the innate immune system in animals and insects. Such peptides are currently produced by extracting them from the host organism or by solid phase peptide synthesis; both techniques are expensive and produce low yields. Recombinant DNA technology opens a window to produce these peptides inexpensively and in large quantities utilizing E. coli expression systems. Two antimicrobial peptides, indolicidin and PGQ, were the focus of this work. They are short amphipathic alpha helical antimicrobial peptides that target a broad range of microorganisms. Genes encoding multimers of indolicidin, PGQ and a hybrid of indolicidin:PGQ were placed into protein expression vectors pET32a+ and pET43.1a+, for peptide production in E. coli. A combination of multimerization and the use of a fusion protein were utilized to mask the toxicity of these peptides in E. coli. The multimerized peptide fusion construct was purified using Ni/NTA affinity chromatography. Methionine residues flanking each monomeric unit were utilized to enable cleavage of the multimerized protein and liberating a biologically active peptide. A Trx:indolicidin trimer fusion was produced in the greatest yield of all constructs investigated. Upon cyanogen bromide cleavage, a band corresponding to the theoretical molecular weight of an indolicidin monomer was observed with SDS-PAGE. Antimicrobial activity of monomeric recombinant indolicidin was tested resulting in zones of clearing. Overall the results indicate that multimerizing antimicrobial peptide genes can potentially produce a larger quantity of peptide per bacterial cell. These studies suggest that multimerization of antimicrobial peptide genes represents a means to control in vivo toxicity of the recombinant peptides and increase production relative to single gene fusions.

The goal of this project was to clone and express the antimicrobial peptide protegrin 1 (PG-1). Initially a yeast system was chosen but was discarded due to technical difficulties. Invitrogen's bacterial T7 expression system was chosen next to express the peptide. PG-1 expression was verified by anti-his immunoblot and then the peptide was purified by IMAC. Its activity was verified using a Bacillus subtillis radial diffusion assay.